Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction.
Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction.
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发表时间：
1986-01
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通讯作者：
T. Shimada;K. Misono;F. Guengerich
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作者：
T. Shimada;K. Misono;F. Guengerich
根据美芬妥英4-羟化酶活性，从人肝微粒体中纯化了两种形式的细胞色素P-450（P-450），命名为P-450 MP-1和P-450 MP-2，使其电泳均一。纯化的P-450 MP-1和P-450 MP-2含有12-17 nmol P-450/mg蛋白质，表观单体分子量分别为48，000和50，000。P-450 MP-1和P-450 MP-2被认为是非常相似的蛋白质，通过在正辛基氨基琼脂糖4 B，羟基磷灰石，和DEAE-和CM-纤维素柱上的色谱行为，光谱特性，氨基酸组成，肽图谱，双免疫扩散分析，免疫抑制和N-末端氨基酸序列来判断。肝脏RNA的体外翻译产生了与P-450 MP-1或P-450 MP-2迁移的多肽，这取决于每个样品中的形式，表明两种P-450是从不同的mRNA翻译的。当与NADPH-细胞色素-P-450还原酶和L-α-二月桂酰-sn-甘油基-3-磷酸胆碱复合时，P-450 MP-1和P-450 MP-2在微粒体中对美芬妥英4-羟基化的转换数明显高于P-450。将纯化的大鼠或人细胞色素b5添加到重构系统中引起羟基化活性的显著增加;当细胞色素b5与P-450的摩尔比为3倍时获得最大刺激。兔抗人细胞色素b5抑制人肝微粒体中的NADH-细胞色素c还原酶和S-美芬妥英4-羟化酶活性。在细胞色素b5的存在下，S-美芬妥英的Km值为1.25 mM，所有5种纯化的细胞色素P-450制剂，Vmax值为0.8-1.25 nmol的4-羟基产物形成每分钟/nmol的P-450。P-450 MP是一种相对选择性的P-450形式，可以很好地代谢取代的乙内酰脲。在人肝微粒体中，由纯化的P-450 MP-1和P-450 MP-2制剂催化并由抗P-450 MP抑制的反应包括S-美芬妥英4-羟基化、S-尼凡诺4-羟基化、S-美芬妥英N-脱甲基化和二苯基乙内酰脲4-羟基化。因此，至少两种非常相似的人P-450形式参与S-美芬妥英4-羟基化，这是一种显示遗传多态性的活性。
Two forms of cytochrome P-450 (P-450), designated P-450MP-1 and P-450MP-2, were purified to electrophoretic homogeneity from human liver microsomes on the basis of mephenytoin 4-hydroxylase activity. Purified P-450MP-1 and P-450MP-2 contained 12-17 nmol of P-450/mg of protein and had apparent monomeric molecular weights of 48,000 and 50,000, respectively. P-450MP-1 and P-450MP-2 were found to be very similar proteins as judged by chromatographic behavior on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE- and CM-cellulose columns, spectral properties, amino acid composition, peptide mapping, double immunodiffusion analysis, immunoinhibition, and N-terminal amino acid sequences. In vitro translation of liver RNA yielded polypeptides migrating with P-450MP-1 or P-450MP-2, depending upon which form was in each sample, indicating that the two P-450s are translated from different mRNAs. When reconsituted with NADPH-cytochrome-P-450 reductase and L-alpha-dilauroyl-sn-glyceryo-3-phosphocholine, P-450MP-1 and P-450MP-2 gave apparently higher turnover numbers for mephenytoin 4-hydroxylation than did the P-450 in the microsomes. The addition of purified rat or human cytochrome b5 to the reconstituted system caused a significant increase in the hydroxylation activity; the maximum stimulation was obtained when the molar ratio of cytochrome b5 to P-450 was 3-fold. Rabbit anti-human cytochrome b5 inhibited NADH-cytochrome-c reductase and S-mephenytoin 4-hydroxylase activities in human liver microsomes. In the presence of cytochrome b5, the Km value for S-mephenytoin was 1.25 mM with all five purified cytochrome P-450s preparations, and Vmax values were 0.8-1.25 nmol of 4-hydroxy product formed per min/nmol of P-450. P-450MP is a relatively selective P-450 form that metabolizes substituted hydantoins well. Reactions catalyzed by purified P-450MP-1 and P-450MP-2 preparations and inhibited by anti-P-450MP in human liver microsomes include S-mephenytoin 4-hydroxylation, S-nirvanol 4-hydroxylation, S-mephenytoin N-demethylation, and diphenylhydantoin 4-hydroxylation. Thus, at least two very similar forms of human P-450 are involved in S-mephenytoin 4-hydroxylation, an activity which shows genetic polymorphism.